Abstract

The use of prebiotics has increased in human and animal nutrition because of
their productive performance and health benefits (Van Loo and Van Craeynest
2008). The objective of this study was to evaluate the prebiotic effect of
Agave fourcroydes agavins from Cuba in pigs in the growth stage. One
hundred and five (Yorkshire-Landrace x L35 hybrid) pigs of 33 days of age
and average initial BW of 8.0±0.95 kg were used in the trial. They were fed in
parallel form with a standard diet or diets supplemented with 0.25% or 0.50% of agavins from Agave fourcroydes
from Cuba for 42 days. The productivity, hematological, microbial indicators,
gastrointestinal organ weights were determined.

Supplementation with agavins did not
influence the growth performance of the animals. Dietary Agave fourcroydes
agavins increased relative organ weight of the small intestine, the cecum,
spleen, thymus and colon and healthy lungs, with
respect to the standard diet. The lymphocyte cells increased in
serum, while pH indicators and coliforms in the colon decreased in the
groups that consumed agavins. In conclusion, the inclusion of agavins from Agave fourcroydes
in pig diets induced a prebiotic response and this constitutes a promising
alternative with potential use in animal nutrition.

Key words: antibiotics, carbohydrates, imune response, microorganisms

Introduction

Pig production in Cuba is characterized by high productive intensity, in which
animals are subjected to different stress situations that cause imbalances
in the intestinal microbiota, development of pathogenic microorganisms,
immuno-suppression and inefficient feed conversion (Sulabo et al 2010).

To counteract these difficulties, for several decades, antibiotics were used
as additives to promote animal growth. However, the indiscriminate use of
these products caused residual effects in food, the development of resistant
pathogenic strains and damage to the ecological balance of the
gastrointestinal biota (Brunser et al 2006). That is why, from the year 2006,
in the European Union and some other countries, the use of antibiotics for
these purposes was prohibited (Van Loo and Vancraeynest 2008).
Therefore, the scientific community and the industrial livestock sector
face the challenge of finding new, safe and innocuous additives.

In this sense, in the nutrition of monogastric animals, the use of prebiotic
additives is increasing, due to the beneficial effects they exert on health
and productive behavior (Chuanlai et al 2005; Janssens and Van Loo 2006).

Prebiotics are defined as a substrate that is selectively utilized by host
microorganisms conferring a health benefit (Gibson et al 2017). Agavins from the agave plant
have been used as prebiotics in different investigations (García-Vieyra
et al 2014; García-Curbelo et al 2015a).

The objective of this study was
to investigate the effects of Agave fourcroydes
agavins as a prebiotic in pigs.

Materials and methods

Agavins

Agavines are carbohydrates extracted from the stems of
Agaves. In this study we used the agavins from Agave fourcroydes which contain oligosaccharides with a DP < 10,
linkages of the type
β
(2-1),
β (2-6), and branches of the neo type (García-Curbelo et al 2015b).

Animals, management and treatments

One hundred and five (Yorkshire-Landrace x L35 hybrid) pigs of 33 days of age
and average initial BW of 8.0±0.95 kg were used in the trial. At the beginning
of the experiment, pigs were randomly distributed into three dietary treatments
by a randomized complete block design. There were eight replications (pens) per treatment with 5 pigs per
pen. The experiment
lasted 75 days.

The treatments were a maize–soybean standard diet supplemented with 0, 0.25
and 0.50% of Agave fourcroydes agavins (STD; 0.25% AF-C; 0.50%
AF-C). The composition of the diets was according to the nutrient
requirements set by the
National Research Council (1998). The diets were free of antibiotics and
coccidiostats. Throughout all the experimental period, the pigs had
ad libitum access to feed and water. All experimental protocols were
approved by the Ethics Committee of the Institute of Animal Science., Cuba.

Sampling and measurements

Body weight (BW), feed intake (FI), body weight gain (BWG) and feed
conversion ratio (FCR) were determine at the end of ethe xperiment period.
Mortality rate was recorded on a daily basis.

At the end of the experiment three animals were selected according to the
average BW within each group and sacrificed. The organs: stomach, small
intestine, cecum, colon, liver, heart, pancreas, spleen, thymus, healthy
lungs were separated, individually weighed, and expressed as percentage of
live body weight.

At the end of the experiment, one pig was randomly chosen from each pen and
blood samples were taken by jugular venipuncture. Blood samples were
centrifuged at 2.000×g at 4°C for 30 min and serum was separated.
Hemoglobin, hematocrit, leukocytes, lymphocytes, and monocytes were determined
according to the blood biochemistry laboratory of the Institute of Animal
Science procedures.

Colon contents were collected and the pH of the contents measured (pH meter
AB150). For microbiological examination of colon contents the visual
counts of total anaerobic bacteria, lactobacilli and coliforms were made.
Selective media were used to identify total anaerobic bacteria (NRF) (Cadwell
and Bryant 1966), lactic acid producing bacteria (LAB) (MRS) (Rogosa et al
1951), and coliforms in red-violet agar and bile (BioCen 2001),. The
bacterial counts from the samples were performed according to procedures
described by Hungate (1969).

Statistical analyses

Results are expressed as mean values with their standard errors. Differences
between the groups were evaluated using one-way ANOVA followed by simple classification
analysis . Duncan’s (1955), multiple-range test was employed in the
apprppropriate cases through the use of
INFOSTAT (Balzarini et al 2001). Significant differences were considered at
p< 0.05.

Results

Supplementation with agavins did not influence the growth performance of the
animals (Table 1). However, in the pigs that consumed agavins, the relative
weights of the small intestine in AF-C 0.50% treatment with contein of 6.5 % as
compared with the CTL group. In the agavins groups (AF-C 0.25%; AF-C
0.50%), there were increases in relative weights of the cecum contents of 22% and 26%;
in colon
contents of 5% and 6.17%; in spleen contentts 6.83% and 13.04% and thymus
contents of 42% and 61%, as compared with the control group (Table 2).

Table 1.
Effect of Agave fourcroydes agavins on production
indicators of growing pigs

Indicators

STD

AF-C0.25 %

AF-C0.50 %

SEM

Initial weight, kg

8.10

8.10

8.15

0.05

Final weight, kg

21.1

21.5

21.4

0.30

Feed intake, kg

27.2

26.9

26.8

0.28

Weight gain, kg

12.9

13.3

13.3

0.29

Feed conversion

2.10

2.01

2.02

0.04

Mortality, pigs

2

3

1

Also, the weight of healthy lungs in the agavins group increased with respect
to the control by 16.8 and 35.6%, for the AF-C 0.25% and AF-C 0.50%,
treatments, respectively. The other
gastrointestinal organs were not affected by the administration of agavins.

Table 2.
Effect of Agave fourcroydes agavins on organ weights

Organs, g/kg LW

STD

AF-C0.25 %

AF-C0.50 %

SEM

Stomach

10.0

9.29

9.45

0.25

Small intestine

38.6a

40.ab

41.2b

0.47*

Two cecum

2.24a

2.75b

2.84b

0.03*

Colon

1.62a

1.70b

1.72b

0.01**

Liver

22.8

23.3

21.9

0.65

Heart

3.79

4.14

3.74

0.15

Pancreas

0.18

0.23

0.19

0.01

Spleen
Thymus

1.61a0.59a

1.72b0.84b

1.82b0.95b

0.02*
0.05*

Healthy lungs (%)

61.3a

78.1b

96.9c

1.70**

Probability of significance: * P < 0.05, **P < 0.01. Values followed by
different superscripts in each row are different at p <0.05).

The hematological indicators (table 3) are in the range established as normal values ​​for
the species and category studied, only the lymphocytes increased with the addition of agavins with respect
to the STD groups by 50% and 53%, respectively.

Table 3.
Effect of agavins on hematological indicators sampled

Indicators

CTL

AF-C0.25 %

AF-C0.50 %

SEM

Hemoglobin, g/dL

10.0

11.0

11.3

0.39

Hematocrit, %

30.0

33.0

34.0

0.65

Leukocytes, x10-9

11.5

11.3

11.2

0.43

Lymphocytes, %

0.52a

0.78b

0.80b

0.04*

Monocytes, %

0.03

0.03

0.03

0.01

Probability of significance: * P < 0.05. Values followed by different
superscripts in each row are significantly different (P < 0.05).

A decrease in colon pH ws observed in the pigs fed agavins. There was
also a decrease in the population of coliforms. There were no
differences in the other microbial groups analyzed.

Table 4.
Effect of agavins on pH and microorganisms in the colonic
content

Indicators

STD

AF-C0.25 %

AF-C0.50 %

± ES

Total bacteria, x10-9

3.08
(31.1)

1.99
(8.39)

1.65
(9.50)

0.39
P=0.05

Lactobacillus, x10-7

4.68
(110)

4.30
(85.0)

3.98
(69.3)

0.26
P=0.20

Coliformes, x10-6

3.19 b(26.0)

1.50 a(5.28)

1.92 a(7.45)

0.21*
P=0.05

pH

5.97a

5.41b

5.39b

0.05*

Values
followed by different superscripts in each row are
different at p< 0.05

Discussion

No differences were observed in productive traits of growing pigs when
agavins of Agave fourcroydes were included in the diet. In the scientific
literature there are variable results with respect to the effect that these
compounds exert on the productive performance. It is to be expected that the
action of these compounds will be manifested to a greater extent when the animals are
subjected to stressful conditions (Chuanlai et al 2005; Shim 2005). In
evaluating the use of prebiotics it is necessary to take into account that their
effects depend on the type of compound, the dose, the animal species, the
conditions of exploitation, among others (Piva and Rossi 1999).

The intake of agavins increased cecum and colon weight, at both levels of
intake. Agavins β links are not degraded by the digestive enzymes of
the host in the upper gastrointestinal tract, reaching the large intestine
unchanged, a site with high microbial populations, where beneficial bacteria
use these compounds as an energy source, consequently increasing generation
of short chain fatty acids (SCFA), which may result in an increase in crypt depth, cell density and cell
proliferation (Santos et al 2006).

The pH in the colon was reduced in the pigs
that consumed agavins. The SCFA were not determined in this study, but it us
likely they were the cause of the lower
pH and other positive effects in the gastrointestinal tract.

The dose differences of these products might influence their degree of fermentation by
the microbiota. In previous studies, it was reported that when agavins of
Agave forucroydes were added to mice diets the concentration of butyrate in the cecal digesta increased (García-Curbelo et al 2015a).
Rehman et al (2008) reported that adding inulin to broiler diets increased the butyrate concentration in
the cecum. Butyrate provides
energy for epithelial growth (Topping and Clifton 2001) and is involved directly or
indirectly on various mechanisms such as cellular differentiation, growth,
permeability, and gene expression (Mroz et al 2006).

The increased production of SCFA as
a result of administration of prebiotics could result in increased intestinal
acidity which may also contribute to the suppression of pathogens in the pig gut
(Wall et al 2012). In this study with the use of agavins a decrease in coliforms
was achieved.

An important result was the increase in the percentage of healthy lungs
found in the groups that consumed agavines. There was also a lower incidence
of respiratory disorders which are very frequent in growing pigs in tropical
countries (Cabrera et al 2006). These results make it possible to avoid the use
of antibiotics in pig production, and to increase the number of healthy
animals.

The immunomodulatory properties of prebiotics have been described in different researches
(Dubert-Ferrandon et al 2008; Madej et al 2015). This study showed that the agavins increased
relative weight of spleen and thymus. These organs are related to the immune response
and they are very important during the first stage of the animals’ life (Onifades 1997).

The reasons why supplementation with agavins enhanced the immune system of pigs were not investigated
in this study, but prebiotics are known to prevent the colonization of pathogens
by adhering to,
and blocking of the intestinal surface. This stimulates the intestinal immune-competent cells,
associated to the lymphoid issue; prebiotics notify the immune system through
the activation of macrophages
and favor high levels of immunoglobulin (local and systemic) (Seifert and Watz 2008;
Vidanarachchi et al 2013).

The hematological indicator values in this study agree with those reported as normal for the species
and animal category (Sotolongo et al 2006; Gélvez 2009). Only the lymphocytes were increased
with the addition of agavins with respect to the control, which may be related to the
stimulation of the immune system exerted by these compounds.

Conclusions

The inclusion of agavins from Agave fourcroydes in the diet
provoked a prebiotic response on the physiological status of the pigs in this
study.

Shim SB 2005
Effects of prebiotics, probiotics and synbiotics in the diet of young pigs
ph.d. Thesis, Animal Nutrition Group, Wageningen Institute of Animal
Sciences, Wageningen University and Research Centre, Wageningen, The
Netherlands With summaries in English and Dutch ISBN 90-8504-193-7.